The present invention relates to compositions and methods for detecting the pathogenic yeast species, Candida albicans and Candida dubliniensis. More specifically, the invention relates to hybridization probes and accessory polynucleotides having specificity for the ribosomal nucleic acids of these species.
The yeast Candida albicans (C. albicans) is one of the most common fungal pathogens that infect humans. Although it is part of the normal flora of the mucous membranes in the respiratory, gastrointestinal and female genital tracts, this opportunistic pathogen may gain dominance in these locations and result in disease conditions. Indeed, while C. albicans infections of otherwise healthy individuals are rarely fatal, infections can lead to life-threatening conditions in persons having comprised immune systems such as, for example, AIDS patients, cancer patients undergoing chemotherapy, and organ transplant patients receiving immunosuppressive drugs. Disseminated (systemic) candidiasis is most prevalent in immunosuppressed individuals and is usually established following passage of the organism across the mucosal epithelium into the bloodstream. Once in the bloodstream, this organism can cause thrombophlebitis, endocarditis, or infection of the eyes. Further, C. albicans can invade virtually any organ or tissue when introduced intravenously, e.g., via tubing, needles or narcotics abuse.
Candida is a heterogeneous genus of yeast. At least six Candida species have been implicated as human pathogens, although the majority of Candida infections are caused by C. albicans and C. tropicalis. Since proper diagnosis and early treatment of an infection by C. albicans provides a clear advantage with respect to medical treatment, it is highly desirable to have available methods of detecting a wide range of different strains of this organism.
The discovery of the novel opportunistic pathogen Candida dubliniensis has added to the range of organisms responsible for disease in humans (see Sullivan et al., Microbiology 141:1507 (1995)). Isolates of this yeast species have been primarily recovered from the oral cavities of HIV-infected patients, both with and without clinical symptoms of oral candidiasis. However, C. dubliniensis has also been recovered from the oral cavities of asymptomatic and symptomatic immunocompetent individuals, although to a much lesser extent. There are also a few reports of the isolation of C. dubliniensis from sputum, blood, fecal and vaginal specimens. In a study reported by Odds et al., in J. Clin. Microbiol. 36:2869 (1998), about 2% of yeasts originally identified as C. albicans were reidentified as C. dubliniensis on the basis of DNA fingerprinting.
It is well established that two single strands of deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d) or ribonucleic acid (xe2x80x9cRNAxe2x80x9d) can associate or xe2x80x9chybridizexe2x80x9d with one another to form a double-stranded structure having two strands held together by hydrogen bonds between complementary base pairs. The individual strands of nucleic acid are formed from nucleotides that comprise the bases: adenine (A), cytosine (C), thymine (T), guanine (G), uracil (U) and inosine (I). In the double helical structure of nucleic acids, the base adenine hydrogen bonds with the base thymine or uracil, the base guanine hydrogen bonds with the base cytosine and the base inosine hydrogen bonds with adenine, cytosine or uracil. At any point along the chain, therefore, one may find the classical xe2x80x9cWatson-Crickxe2x80x9d base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may also find A:G, G:U and other xe2x80x9cwobblexe2x80x9d or mismatched base pairs in addition to the traditional (xe2x80x9ccanonicalxe2x80x9d) base pairs.
A double-stranded nucleic acid hybrid will result if a first single-stranded polynucleotide is contacted under hybridization-promoting conditions with a second single-stranded polynucleotide having a sufficient number of contiguous bases complementary to the sequence of the first polynucleotide. DNA/DNA, RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.
Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with the nucleic acid sequence that is to be detected (xe2x80x9ctarget sequencexe2x80x9d). Probes commonly are labeled with a detectable moiety such as a radioisotope, an antigen or a chemiluminescent moiety.
Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and 5,681,698. These references also describe methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These procedures require probes that are sufficiently complementary to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. According to the method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and then incubated under specified hybridization conditions. Conventionally, but not necessarily, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and quantitate the amount of labeled probe that has formed duplex structures in order to detect the presence of rRNA contained in the test sample.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding homologs of the procaryotic 5S, 16S and 23S rRNA molecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA which are substantially similar to the prokaryotic molecules. Probes for detecting specifically targeted rRNA subsequences in particular organisms or groups of organisms in a sample have been described previously. These highly specific probe sequences advantageously do not cross react with nucleic acids from any other bacterial species or infectious agents under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect a broad range of Candida albicans strains and Candida dubliniensis in a highly specific manner.
A first aspect of the invention relates to an oligonucleotide that has a length of up to 100 nucleotides and a sequence that includes at least 17 contiguous nucleotides from SEQ ID NO:6 or the complement thereof. The sequence of the oligonucleotide may include any one of SEQ ID NO: 1 or the complement thereof, SEQ ID NO:2 or the complement thereof, SEQ ID NO:3 or the complement thereof, SEQ ID NO:4 or the complement thereof, or SEQ ID NO:5 or the complement thereof. In each of these cases, rather than having a length of up to 100 nucleotides, the oligonucleotide may have a length of up to only 60 nucleotides. In certain embodiments, the oligonucleotide is made of DNA. In certain other embodiments of the invention the sequence of the oligonucleotide is given precisely by of any one of SEQ ID NO: 1 or the complement thereof, SEQ ID NO:2 or the complement thereof, SEQ ID NO:3 or the complement thereof, SEQ ID NO:4 or the complement thereof, and SEQ ID NO:5 or the complement thereof. When this is the case, the oligonucleotide optionally may include a detectable label. In a highly preferred embodiment, the sequence of the oligonucleotide is given by either of SEQ ID NO:1 or SEQ ID NO:4. In an even more highly preferred embodiment, the oligonucleotide which has the sequence of either SEQ ID NO:1 or SEQ ID NO:4 further includes a detectable label. Examples of detectable labels include chemiluminescent labels, such as acridinium esters.
A second aspect of the invention relates to a composition that can be used for detecting the nucleic acids of a yeast that is either C. albicans or C. dubliniensis. This composition includes an oligonucleotide probe having a length of up to 100 nucleotide bases and a sequence that includes at least 17 contiguous nucleotides from SEQ ID NO:6 or the complement thereof. In certain instances, rather than having a length of up to 100 nucleotides, the length of the oligonucleotide probe is up to only 60 nucleotides. The probes in the invented composition may conveniently be made of DNA. In certain preferred embodiments of the invented oligonucleotide probe there is included a detectable label, which is a chemiluminescent label or a radiolabel, and the length of the probe is no longer than 60 nucleotides. When the sequence of the oligonucleotide probe is given precisely by either SEQ ID NO:1 or SEQ ID NO:4, and further when the probe includes a detectable label, the detectable label may be a chemiluminescent label or a radiolabel. As a particular example, the detectable label is a chemiluminescent label, and this chemiluminescent label is an acridinium ester. Alternatively, when the sequence of the oligonucleotide probe is given precisely by either SEQ ID NO:1 or SEQ ID NO:4, and further when the probe includes a detectable label, there may be included in the composition at least one helper oligonucleotide. According to one highly preferred embodiment of the invention, the sequence of the oligonucleotide probe is given by SEQ ID NO:1, and the helper oligonucleotide is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3. According to another highly preferred embodiment, the sequence of the oligonucleotide probe is given by SEQ ID NO:4, and the helper oligonucleotide is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
A third aspect of the invention relates to a method of determining whether a test sample includes an organism that is either C. albicans or C. dubliniensis. This method includes a first step for providing to the test sample a probe composition that includes an oligonucleotide probe having a length of up to 100 nucleotide bases and a sequence that includes at least 17 contiguous nucleotides from SEQ ID NO:6 or the complement thereof. A second step in the method involves hybridizing under a high stringency condition any nucleic acid that may be present in the test sample with the probe composition to form a probe:target duplex. Finally, there is a step for detecting the probe:target duplex. When such a duplex is detected it indicates that an organism which is either C. albicans or C. dubliniensis is present in the test sample. This method may be carried out using oligonucleotide probes that have sequences given by either SEQ ID NO:1 or SEQ ID NO:4. In this instance, it is possible that the test sample may include yeast cells, and before the above-indicated first step there is a preliminary step for releasing nucleic acid from any yeast cells that may be present in the test sample. Alternatively, the test sample may begin as a lysate. Examples of useful high stringency hybridization conditions that can be used in the second step of the method include either: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, 1 mM each of EDTA and EGTA; or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. When the sequence of the oligonucleotide probe consists of either SEQ ID NO:1 or SEQ ID NO:4, it is preferred that the oligonucleotide probe also includes a detectable label. In one embodiment of the invented method, this detectable label is an acridinium ester, and the xe2x80x9cdetectingxe2x80x9d step involves performing luminometry to detect any of the probe:target duplex. In another embodiment of the invented method, the probe composition further includes at least one helper oligonucleotide. For example, it is highly preferred for the sequence of the oligonucleotide probe to be given by SEQ ID NO:1, and further that at least one helper oligonucleotide has a sequence given by SEQ ID NO:2 or SEQ ID NO:3. Alternatively, it is highly preferred for the sequence of the oligonucleotide probe to be given by SEQ ID NO:4, and further that at least one helper oligonucleotide has a sequence given by SEQ ID NO:2 or SEQ ID NO:5.
Definitions
As used herein, the following terms have the given meanings unless expressly stated to the contrary.
A xe2x80x9cnucleotidexe2x80x9d is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2xe2x80x2-deoxyribose. The sugar of a 5xe2x80x2-nucleotide contains a hydroxyl group (xe2x80x94OH) at the 5xe2x80x2-carbon-5 position. The term also includes analogs of naturally occurring nucleotides and particularly includes analogs having a methoxy group at the 2xe2x80x2 position of the ribose (OMe). As used herein, methoxy oligonucleotides containing xe2x80x9cTxe2x80x9d residues have a methoxy group at the 2xe2x80x2 position of the ribose moiety, and a uracil at the base position of the nucleotide. When particularly specified as xe2x80x9cOMeTxe2x80x9d it is meant that the base position of the nucleotide is occupied by a thymine residue.
A xe2x80x9cnon-nucleotide unitxe2x80x9d is a unit which does not significantly participate in hybridization of a polymer. Such units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and would exclude units having as a component one of the five nucleotide bases or analogs thereof.
An xe2x80x9coligonucleotidexe2x80x9d is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about 10 to about 100 nucleotides in length. The sugar groups of the nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may by joined by linkages such as phosphodiester linkages, modified linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleotide sequence. Modified linkages include those in which a standard phosphodiester linkage is replaced with a different linkage, such as a phosphorothioate linkage, a methylphosphonate linkage, or a neutral peptide linkage. Nitrogenous base analogs also may be components of oligonucleotides in accordance with the invention.
A xe2x80x9ctarget nucleic acidxe2x80x9d is a nucleic acid comprising a target nucleic acid sequence.
A xe2x80x9ctarget nucleic acid sequence,xe2x80x9d xe2x80x9ctarget nucleotide sequencexe2x80x9d or xe2x80x9ctarget sequencexe2x80x9d is a specific deoxyribonucleotide or ribonucleotide sequence that can be hybridized by an oligonucleotide.
An xe2x80x9coligonucleotide probexe2x80x9d is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a detectable hybrid probe:target duplex under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions. Non-complementary sequences, such as promotor sequences, restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes. An oligonucleotide probe optionally may be labeled with a detectable moiety such as a radioisotope, a fluorescent moiety, a chemiluminescent moiety, an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence. Oligonucleotide probes are preferred to be in the size range of from 10 to 100 nucleotides in length.
A xe2x80x9cdetectable moietyxe2x80x9d is a molecule attached to, or synthesized as part of, a nucleic acid probe. This molecule should be uniquely detectable and will allow the probe to be detected as a result. These detectable moieties are often radioisotopes, chemiluminescent molecules, enzymes, haptens, or even unique oligonucleotide sequences.
A xe2x80x9chybridxe2x80x9d or a xe2x80x9cduplexxe2x80x9d is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases.
xe2x80x9cHybridizationxe2x80x9d is the process by which two complementary strands of nucleic acid combine to form a double-stranded structure (xe2x80x9chybridxe2x80x9d or xe2x80x9cduplexxe2x80x9d).
xe2x80x9cComplementarityxe2x80x9d is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) ordinarily complements thymine (T) or Uracil (U), while guanine (G) ordinarily complements cytosine (C).
xe2x80x9cMismatchxe2x80x9d refers to any pairing, in a hybrid, of two nucleotides which do not form canonical Watson-Crick hydrogen bonds. In addition, for the purposes of the following discussions, a mismatch can include an insertion or deletion in one strand of the hybrid which results in an unpaired nucleotide(s).
The term xe2x80x9cstringencyxe2x80x9d is used to describe the temperature and solvent composition existing during hybridization and the subsequent processing steps. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid. Exemplary high stringency conditions are provided in the working Examples.
The term xe2x80x9cprobe specificityxe2x80x9d refers to a characteristic of a probe which describes its ability to distinguish between target and non-target sequences.
The term xe2x80x9cvariable regionxe2x80x9d refers to a nucleotide polymer which differs by at least one base between the target organism and non-target organisms contained in a sample.
A xe2x80x9cconserved regionxe2x80x9d is a nucleic acid subsequence which is not variable between at least two different polynucleotides.
The term xe2x80x9csequence divergencexe2x80x9d refers to a process by which nucleotide polymers become less similar during evolution.
The term xe2x80x9csequence convergencexe2x80x9d refers to a process by which nucleotide polymers become more similar during evolution.
xe2x80x9cTmxe2x80x9d refers to the temperature at which 50% of the probe is converted from the hybridized to the unhybridized form.
A xe2x80x9chelper oligonucleotidexe2x80x9d is an oligonucleotide that binds a region of a target nucleic acid other than the region that is bound by an oligonucleotide probe. Helper oligonucleotides impose new secondary and tertiary structures on the targeted region of the single-stranded nucleic acid so that the rate of binding of the oligonucleotide probe is accelerated. Although helper oligonucleotides are not labeled with a detectable label when used in conjunction with labeled oligonucleotide probes, they facilitate binding of labeled probes and so indirectly enhance hybridization signals.
The phrases xe2x80x9cconsist essentially ofxe2x80x9d or xe2x80x9cconsisting essentially ofxe2x80x9d means that the oligonucleotide has a nucleotide sequence substantially similar to a specified nucleotide sequence. Any additions or deletions are non-material variations of the specified nucleotide sequence which do not prevent the oligonucleotide from having its claimed property, such as being able to preferentially hybridize under high stringency hybridization conditions to its target nucleic acid over non-target nucleic acids.
One skilled in the art will understand that substantially corresponding probes of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes of the present invention substantially correspond to a nucleic acid sequence if these percentages are from 100% to 80% or from 0 base mismatches in a 10 nucleotide target sequence to 2 bases mismatched in a 10 nucleotide target sequence. In preferred embodiments, the percentage is from 100% to 85%. In more preferred embodiments, this percentage is from 90% to 100%; in other preferred embodiments, this percentage is from 95% to 100%.
By xe2x80x9csufficiently complementaryxe2x80x9d or xe2x80x9csubstantially complementaryxe2x80x9d is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.
By xe2x80x9cnucleic acid hybridxe2x80x9d or xe2x80x9cprobe:target duplexxe2x80x9d is meant a structure that is a double-stranded, hydrogen-bonded structure, preferably 10 to 100 nucleotides in length, more preferably 14 to 50 nucleotides in length. The structure is sufficiently stable to be detected by means such as chemiluminescent or fluorescent light detection, autoradiography, electrochemical analysis or gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.
By xe2x80x9cnegative sensexe2x80x9d is meant a nucleic acid molecule perfectly complementary to a reference (i.e., sense) nucleic acid molecule.
xe2x80x9cRNA and DNA equivalentsxe2x80x9d refer to RNA and DNA molecules having the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar groups (i.e., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents do not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence.
By xe2x80x9cpreferentially hybridizexe2x80x9d is meant that under high stringency hybridization conditions oligonucleotide probes can hybridize their target nucleic acids to form stable probe:target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probe:non-target hybrids (that would indicate the presence of non-target nucleic acids from other organisms). Thus, the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one skilled in the art to accurately detect the presence of C. albicans and/or C. dubliniensis and distinguish these species from other organisms. Preferential hybridization can be measured using techniques known in the art and described herein.
A xe2x80x9ctarget nucleic acid sequence regionxe2x80x9d refers to a nucleic acid sequence present in the nucleic acid of an organism or a sequence complementary thereto, which is not present in the nucleic acids of other species. Nucleic acids having nucleotide sequences complementary to a target sequence may be generated by target amplification techniques such as polymerase chain reaction (PCR) or transcription mediated amplification (e.g., Kacian and Fultz, Nucleic Acid Sequence Amplification Methods, U.S. Pat. No. 5,824,518).
Herein we disclose preferred target nucleotide sequences for oligonucleotide probes and helper oligonucleotides that can be used to detect and identify rRNA or rDNA of Candida albicans and Candida dubliniensis without substantially detecting ribosomal nucleic acids of other microorganisms or humans. Highly preferred polynucleotide probes and accessory helper oligonucleotides that specifically detect C. albicans and C. dubliniensis are particularly disclosed. Probes which are complementary to particular rRNA sequences of the 28S rRNA advantageously are capable of distinguishing these species from the known phylogenetically nearest neighbors.
In addition to having nucleic acid sequences that permit hybridization to the ribosomal RNA (rRNA) or DNA (rDNA) sequences of C. albicans and C. dubliniensis, the oligonucleotides of the invention are at least 90% complementary, preferably perfectly complementary, to at least a portion of the described target sequence region identified by SEQ ID NO:7. The portion is preferably at least 15 nucleotides in length, more preferably at least 17 nucleotides in length, still more preferably at least 23 nucleotides in length, even more preferably at least 25 nucleotides in length, and yet even more preferably at least 34 nucleotides in length.
As indicated above, the invented oligonucleotides are targeted to nucleic acid sequences of C. albicans and C. dubliniensis. These oligonucleotides can be used as probes that preferentially hybridize a nucleic acid target region to form a detectable duplex that indicates the presence of either C. albicans or C. dubliniensis. Alternatively, the invented oligonucleotides can be used as helper oligonucleotides that hybridize a nucleic acid target region under high stringency hybridization conditions, and that can enhance the formation of a duplex between a labeled oligonucleotide probe and its complementary target nucleic acid.
In preferred embodiments, the oligonucleotide probes described herein selectively hybridize nucleic acids from C. albicans and C. dubliniensis over those from other organisms under high stringency hybridization conditions. In some embodiments of the present invention, the oligonucleotide probe comprises a detectable moiety, such as an acridinium ester or a radioisotope.
Preferred methods for detecting the presence of C. albicans and C. dubliniensis include the step of contacting a test sample under high stringency hybridization conditions with an oligonucleotide probe that preferentially hybridizes to a C. albicans and C. dubliniensis target nucleic acid sequence over a nucleic acid sequence of other organisms. Preferably the target nucleic acid sequence has at least 15 contiguous nucleotides, more preferably at least 17 contiguous nucleotides, still more preferably at least 23 contiguous nucleotides, even more preferably at least 25 contiguous nucleotides, and yet even more preferably at least 34 contiguous nucleotides of the sequence given by SEQ ID NO:7. Thus, oligonucleotides useful for hybridizing the rRNA target preferably are up to 100 nucleotides in length, or more preferably up to 60 nucleotides in length, and have at least 15 contiguous nucleotides, more preferably at least 17 contiguous nucleotides, still more preferably at least 23 contiguous nucleotides, even more preferably at least 25 contiguous nucleotides, and yet even more preferably at least 34 contiguous nucleotides contained within the sequence of SEQ ID NO:6. However, other useful probes for hybridizing rDNA are up to 100 nucleotides in length, or more preferably up to 60 nucleotides in length, and have at least at least 15 contiguous nucleotides, more preferably at least 17 contiguous nucleotides, still more preferably at least 23 contiguous nucleotides, even more preferably at least 25 contiguous nucleotides, and yet even more preferably at least 34 contiguous nucleotides of the sequence given by the complement of SEQ ID NO:6.
Introduction and Background
In the development of the invention, rRNA sequences from a collection of related and unrelated organisms were aligned to identify candidate conserved sequences present in the 28S rRNA that could be used to distinguish Candida albicans from other organisms. The rRNA or rDNA sequences of Candida albicans and distant phylogenetic neighbors were aligned to reveal areas of maximum homology. Homologous regions were examined for sequence variation in order to identify rRNA sequences that showed mismatches with other closely and distantly related genera. The sequences deduced as candidate probes according to the methods described below finally were tested against a panel of rRNA standards and rRNA-containing cell lysates to verify their utility as probes under laboratory conditions.
Polynucleotide sequences of rRNAs are most conveniently determined using a dideoxynucleotide sequencing procedure. In this procedure, oligonucleotide primers of about 10-100 bases in length and complementary to conserved regions of rRNA from any of the ribosome subunits can be extended by reverse transcriptase. The resulting DNA extension products can then be sequenced either by chemical degradation or by dideoxynucleotide sequencing (Lane et al., Proc. Natl. Acad. Sci. USA 82: 6955 (1985)). According to another preferred method, genomic sequences encoding the rRNA can also be determined.
The strong interdependence of secondary structure and function of the rRNA molecules is well known. Indeed, evolutionary changes in the primary sequence of the rRNA are effectively restricted such that secondary structure of the molecule will be maintained. For example, if a base is changed on one side of a helix of a rRNA molecule, then a compensating change will be made on the other side of the helix to preserve complementarity (this is referred to as covariance). This relationship allows two very different rRNA sequences to be xe2x80x9calignedxe2x80x9d based on conserved primary sequence and conserved elements of the secondary structure. Once the sequences have been aligned, it becomes possible to identify conserved and variable regions of the rRNA sequence.
Variable regions of rRNAs were identified by comparative analysis using published rRNA sequences and sequences that were determined during the development of the present invention. Commercially available software can be used or adapted for the purposes disclosed herein. Since the sequence evolution at each of the variable regions (for example, spanning a minimum of 10 nucleotides) of rRNA is, for the most part, divergent and not convergent, we can confidently design probes based on a few rRNA sequences which differ between the target organism and its phylogenetically closest relatives. Indeed, we have detected sufficient variation between the rRNA sequences of numerous target organisms and their closest phylogenetic relatives in a single sample to permit the design of a probe that can be used according to the methods described below.
Probe Selection Guidelines
The following general guidelines can be used for designing probes having desirable characteristics in accordance with the present invention. Manipulation of one or more of the many factors that influence the extent and specificity of a hybridization reaction can determine the sensitivity and specificity of a particular probe. This is true whether or not the probe is perfectly complementary over the full length of its target polynucleotide sequence. Guidelines for preparing probes useful in connection with the invention now follow.
First, the stability of the probe:target nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long A and T rich sequences, by terminating the hybrids with G:C base pairs and by designing the probe in such a way that the Tm will be appropriate for standard conditions to be employed in the assay. The nucleotide sequence of the probe should be chosen so that the length and %G and %C result in a probe having a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G:C base pairs exhibit greater thermal stability when compared with A:T base pairs. Thus, hybrids involving complementary nucleic acids having a high G:C content will be stable at higher temperatures when compared with hybrids having a lower G:C content.
Ionic strength and temperature conditions at which a hybridization reaction will be conducted also should be considered when designing a probe having a negatively charged backbone, such as would be provided by phosphodiester linkages between nucleotides. It is generally known that hybridization rate increases as ionic strength of the reaction mixture increases. Similarly, the thermal stability of hybrids increases with increasing ionic strength. Conversely, hydrogen bond-disrupting reagents such as formamide, urea, DMSO and alcohols increase the stringency of hybridization. Destabilization of hydrogen bonds by reagents in this class can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Hybridization reactions conducted below the temperature optimum may allow mismatched base sequences to hybridize and can result in reduced probe specificity.
Second, the position at which the probe binds its target polynucleotide should be chosen to minimize the stability of hybrids formed between probe:non-target polynucleotides. This may be accomplished by minimizing the length of perfect complementarity with polynucleotides of non-target organisms, by avoiding G:C rich regions of homology with non-target sequences, and by positioning the probe to span as many destabilizing mismatches as possible. Whether a probe sequence will be useful for detecting only a specific type of organism depends largely on thermal stability differences between probe:target hybrids and probe:non-target hybrids. The differences in Tm should be as large as possible to produce highly specific probes.
The length of the target nucleic acid sequence and the corresponding length of the probe sequence also are important factors to be considered when designing a probe. While it is possible for polynucleotides that are not perfectly complementary to hybridize to each other, the longest stretch of perfectly homologous base sequence will ordinarily be the primary determinant of hybrid stability.
Third, regions of the rRNA which are known to form strong internal structures inhibitory to hybridization of a probe are less preferred as targets. Probes having extensive self-complementarity also should be avoided. As indicated above, hybridization is the association of two single strands of complementary nucleic acid to form a hydrogen bonded double-stranded structure. If one of the two strands is wholly or partially double-stranded, then it will be less able to participate in the formation of a new hybrid. Significantly, all rRNA molecules form very stable intramolecular hybrids.
The rate and extent of hybridization between a probe and its target can be increased substantially by designing the probe such that a substantial portion of the sequence of interest is single-stranded. If the target nucleic acid to be detected is a genomic sequence encoding a rRNA, then that target will naturally occur in a double-stranded form. This is also the case with products of the polymerase chain reaction (PCR). These double-stranded targets are naturally inhibitory to hybridization with a probe. Finally, undesirable intramolecular and intermolecular hybrids can form within a single probe molecule or between different probe molecules if there is sufficient self-complementarity. Thus, extensive self-complementarity in a probe sequence should be avoided.
Preferably, probes useful for carrying out the procedures described below will hybridize under conditions of high stringency. Under these conditions only highly complementary nucleic acid hybrids will form (i.e., those having at least 14 out of 17 bases in a contiguous series of bases being complementary). Hybrids will not form in the absence of a sufficient degree of complementarity. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and non-target nucleic acid. Exemplary high stringency conditions are employed in the Examples presented below.
While oligonucleotide probes of different lengths and base composition may be used for detecting C. albicans and C. dubliniensis, preferred probes in this invention have lengths of up to 100 nucleotides, and more preferably lengths of up to 60 nucleotides. Preferred length ranges for the invented oligonucleotides are from 10 to 100 bases in length, more preferably between 15 and 60 bases in length, or still more preferably between 15 and 50 bases in length. Preferred probes are sufficiently homologous to the target nucleic acid to permit hybridization under high stringency conditions such as those employed in the Examples described below. However, the specific probe sequences described below also may be provided in a nucleic acid cloning vector and still can be used for detecting C. albicans and C. dubliniensis. 
Chemical Structure of Oligonucleotides
All of the oligonucleotides of the present invention may be modified with chemical groups to enhance their performance. Thus, it is to be understood that references to xe2x80x9coligonucleotide probesxe2x80x9d or xe2x80x9chelper oligonucleotidesxe2x80x9d or simply xe2x80x9coligonucleotidesxe2x80x9d embrace polymers of native nucleotides as well as polymers that include at least one nucleotide analog.
Backbone-modified oligonucleotides, such as those having phosphorothioate or methylphosphonate groups, are examples of analogs that can be used in conjunction with oligonucleotides of the present invention. These modifications render the oligonucleotides resistant to the nucleolytic activity of certain polymerases or to nuclease enzymes. Other analogs that can be incorporated into the structures of the oligonucleotides disclosed herein include peptide nucleic acids, or xe2x80x9cPNAs.xe2x80x9d The PNAs are compounds comprising ligands linked to a peptide backbone rather than to a phosphodiester backbone. Representative ligands include either the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine or thiouracil) or artificial bases (e.g., bromothymine, azaadenines or azaguanines, etc.) attached to a peptide backbone through a suitable linker. The PNAs are able to bind complementary ssDNA and RNA strands. Methods for making and using PNAs are disclosed in U.S. Pat. No. 5,539,082. Another type of modification that can be used to make oligonucleotides having the sequences described herein involves the use of non-nucleotide linkers (e.g., Arnold, et al., xe2x80x9cNon-Nucleotide Linking Reagents for Nucleotide Probesxe2x80x9d, U.S. Pat. No. 6,031,091 hereby incorporated by reference) incorporated between nucleotides in the nucleic acid chain which do not interfere with hybridization or the elongation of a primer.
Nucleic Acid Based Methods of Detecting rRNA or rDNA
A composition that includes an oligonucleotide probe, either alone or in combination with one or more helper oligonucleotides, can be used for detecting rRNA or rDNA of either C. albicans or C. dubliniensis in a hybridization assay. Defined oligonucleotides that can be used to practice the invention can be produced by any of several well-known methods, including automated solid-phase chemical synthesis using cyanoethylphosphoramidite precursors (Barone et al., Nucl Acids Res 12:4051 (1984)). Other well-known methods for preparing synthetic oligonucleotides also may be employed.
Essentially any labeling and detection system that can be used for monitoring specific nucleic acid hybridization can be used in conjunction with the probes disclosed herein when a labeled probe is desired. Included among the collection of useful labels are: isotopic labels, enzymes, haptens, linked oligonucleotides, chemiluminescent molecules and redox-active moieties that are amenable to electrochemical detection methods. Standard isotopic labels that can be used to produce labeled oligonucleotides include 3H, 35S, 32p, 125I, 57Co and 14C. When using radiolabeled probes, hybrids can be detected by autoradiography, scintillation counting or gamma counting.
Non-isotopic materials can also be used for labeling oligonucleotide probes. These non-isotopic labels can be positioned internally or at a terminus of the oligonucleotide probe. Modified nucleotides may be incorporated enzymatically or chemically with modifications of the probe being performed during or after probe synthesis, for example, by the use of non-nucleotide linker groups. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.
Indeed, any number of different non-isotopic labels can be used for preparing labeled oligonucleotides in accordance with the invention. Preferred chemiluminescent molecules include acridinium esters of the type disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use in connection with homogenous protection assays, and of the type disclosed by Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connection with assays that quantify multiple targets in a single reaction. The disclosures contained in these patent documents are hereby incorporated by reference. U.S. Pat. No. 5,998,135 discloses yet another method that can be used for labeling and detecting the probes of the present invention using fluorimetry to detect fluorescence emission from lanthanide metal labels disposed on probes, where the emission from these labels becomes enhanced when it is in close proximity to an energy transfer partner. Preferred electrochemical labeling and detection approaches are disclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, and the published International Patent Application No. PCT/US98/12082, the disclosures of which are hereby incorporated by reference. Redox active moieties useful as electrochemical labels in the present invention include transition metals such as Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru.
Those having an ordinary level of skill in the art will appreciate that alternative procedures for detecting nucleic acids of C. albicans and C. dubliniensis using the invented probes can be carried out using either labeled probes or unlabeled probes. For example, hybridization assay methods that do not rely on the use of a labeled probe are disclosed in U.S. Pat. No. 5,945,286 which describes immobilization of unlabeled probes made of peptide nucleic acids (PNAs), and detectably labeled intercalating molecules which can bind double-stranded PNA probe/target nucleic acid duplexes. In these procedures, as well as in certain electrochemical detection procedures, such as those disclosed in published International Patent Application No. PCT/US98/12082 entitled xe2x80x9cDetection of Analytes Using Reorganization Energy,xe2x80x9d published International Patent Application No. PCT/US98/12430 entitled xe2x80x9cElectronic Methods for the Detection of Analytes,xe2x80x9d and in published International Patent Application No. PCT/US97/20014 entitled xe2x80x9cElectrodes Linked Via Conductive Oligomers to Nucleic Acids,xe2x80x9d the oligonucleotide probe is not required to harbor a detectable label.
Acceptability of the final product following synthesis and purification of an oligonucleotide may be verified by any of several procedures. First, polyacrylamide gel electrophoresis can be used to determine the size and purity of the oligonucleotide according to standard laboratory methods (see Molecular Cloning: A Laboratory Manual, Sambrook et al., eds. Cold Spring Harbor Lab Publ., 11.51, (1989)). Alternatively, High Pressure Liquid Chromatography (xe2x80x9cHPLCxe2x80x9d) procedures can be used for this same purpose.
Hybridization between the labeled oligonucleotide probe and target nucleic acid in the procedures described below can be enhanced through the use of unlabeled xe2x80x9chelper oligonucleotidesxe2x80x9d according to the procedure disclosed by Hogan et al., in U.S. Pat. No. 5,030,557 entitled, xe2x80x9cMeans and Methods for Enhancing Nucleic Acid Hybridization.xe2x80x9d As indicated above, helper oligonucleotides bind a region of the target nucleic acid other than the region that is bound by the assay probe. This binding imposes new secondary and tertiary structures on the targeted region of the single-stranded nucleic acid and accelerates the rate of probe binding. Helper oligonucleotides which can be used in combination with labeled oligonucleotide probes of the present invention are preferably 15 to 100 nucleotides in length and have a sequence that includes at least 15 contiguous nucleotides, more preferably at least 25 contiguous nucleotides, or even still more preferably at least 34 contiguous nucleotides contained within the sequence of SEQ ID NO:6 or the complement thereof.
Those having an ordinary level of skill in the art will appreciate that factors affecting the thermal stability of a probe:target hybrid also can influence probe specificity. Accordingly, the melting profile, including the melting temperature (Tm) of probe:target hybrids, should be empirically determined for each probe:target combination. A preferred method for making this determination is described by Arnold et al., in U.S. Pat. No. 5,283,174, entitled xe2x80x9cHomogeneous Protection Assay.xe2x80x9d
One approach for measuring the Tm of a probe:target hybrid involves conducting a hybridization protection assay. According to the method of this assay, a probe:target hybrid is formed under conditions of target excess in a lithium succinate buffered solution containing lithium lauryl sulfate. Aliquots of the xe2x80x9cpreformedxe2x80x9d hybrids are diluted in the hybridization buffer and incubated for five minutes at various temperatures starting below the anticipated Tm (typically 55xc2x0 C.) and increasing in 2-5 degree increments. This solution is then diluted with a mildly alkaline borate buffer and incubated at a lower temperature (for example 50xc2x0 C.) for ten minutes. An acridinium ester (AE) linked to a single-stranded probe is hydrolyzed under these conditions while an acridinium ester linked to a hybridized probe will be relatively xe2x80x9cprotected.xe2x80x9d This procedure is referred to as the hybridization protection assay (xe2x80x9cHPAxe2x80x9d). The amount of chemiluminescence remaining is proportional to the amount of hybrid and is measured in a luminometer by addition of hydrogen peroxide followed by alkali. The data is plotted as percent of maximum signal (usually from the lowest temperature) versus temperature. The Tm is defined as the point at which 50% of the maximum signal remains.
In an alternative approach, the Tm of a probe:target hybrid can be determined using an isotopically labeled probe. In all cases, the Tm for a given hybrid will vary depending on the concentration of salts, detergents and other solutes contained in the hybridization solution. All of these factors influence relative hybrid stability during thermal denaturation (Molecular Cloning: A Laboratory Manual Sambrook et al., eds. Cold Spring Harbor Lab Publ., 9.51 (1989)).
The rate at which a probe hybridizes to its target is a measure of the thermal stability of the target secondary structure in the probe region, and can be can be determined using C0txc2xd measurements. These kinetic measurements of hybridization rate have units of (moles of nucleotide per liter)xc3x97(seconds). Expressed more simply, the C0txc2xd value is the concentration of probe times the half-life of hybridization at that concentration. This value can be determined by hybridizing various amounts of probe to a constant amount of target nucleic acid for a fixed time. For example, 0.05 pmol of target is incubated with 0.012, 0.025, 0.05, 0.1 and 0.2 pmol of probe for 30 minutes. The C0txc2xd may also be determined by hybridizing the target and probe under conditions of target excess and then measuring the increase of duplex formation over time. The amount of hybrid present can be measured using the above-described HPA procedure or by scintillation counting if a radiolabeled probe is used in the procedure. The measured signal, when using AE labeled probe, is then plotted as the log of the percent of maximum Relative Light Units (xe2x80x9cRLUxe2x80x9d) from the highest probe concentration versus probe concentration (moles of nucleotide per liter). The C0txc2xd is graphically determined from the concentration corresponding to 50% of maximum hybridization multiplied by the hybridization time in seconds. These values range from 9xc3x9710xe2x88x926 to 9xc3x9710xe2x88x925 with the preferred values being less than 3.5xc3x9710xe2x88x925. Similar values may be obtained by measuring radioactivity and plotting % hybridization at a given time point vs maximum extent.
In a preferred method of determining whether a biological sample contains rRNA or rDNA that would indicate the presence of C. albicans or C. dubliniensis, nucleic acids may be released from cells by sonic disruption, for example according to the method disclosed by Murphy et al., in U.S. Pat. No. 5,374,522. Other known methods for disrupting cells include the use of enzymes, osmotic shock, chemical treatment, and vortexing with glass beads. Other methods suitable for liberating from microorganisms the nucleic acids that can be subjected to the hybridization methods disclosed herein have been described by Clark et al., in U.S. Pat. No. 5,837,452 and by Kacian et al., in U.S. Pat. No. 5,364,763. Following or concurrent with the release of rRNA, labeled probe may be added in the presence of accelerating agents and incubated at the optimal hybridization temperature for a period of time necessary to a achieve significant hybridization reaction.
An oligonucleotide having the sequence CGGCCATAAAGACCTACCAAGCG (SEQ ID NO:1) was characterized by the criteria of length, Tm and nucleotide sequence and was found to be specific for the rRNA of C. albicans and C. dubliniensis. This polynucleotide, referred to herein as CalB2208 is complementary to a unique segment found in the 28S rRNA of C. albicans and C. dubliniensis. The probe is 23 bases in length, has a Tm of 61.4xc2x0 C. and specifically hybridized rRNA of C. albicans and C. dubliniensis. 
A second oligonucleotide having the sequence CGGCCATAAAGACCTAC (SEQ ID NO:4), which is entirely contained within the sequence of the CalB2208 probe, also was characterized. This latter probe, which is 17 bases in length, had a Tm that was lower than that of the CalB2208 probe, and so permitted hybridization procedures to be carried out using an alternative hybridization temperature condition. Like the CalB2208 probe, the CalB2214 probe also bound the rRNA of C. albicans, but not certain other Candida species.
Both the CalB2208 and CalB2214 probes are illustrations of oligonucleotides that: (1) hybridize the target nucleic acid under high stringency hybridization conditions, (2) have lengths of up to 100 nucleotide bases, and (3) include at least 15 contiguous nucleotides falling within the target region identified by SEQ ID NO:6 or its complement. Other oligonucleotides having these properties are contemplated for use as hybridization assay probes and are embraced by the invention.
Similarly, oligonucleotides having the sequences of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5 are disclosed herein as illustrations of useful helper oligonucleotides. Like the helper oligonucleotides employed in the working Examples herein, other helper oligonucleotides embraced by the invention also have sequences of up to 100 nucleotides in length and further have at least 15, or more preferably at least 25, or still more preferably at least 34 contiguous nucleotides contained within the target region identified by SEQ ID NO:6 or its complement.
As indicated below, the CalB2208 probe hybridized C. albicans rRNA in a manner that was promoted by the use of helper oligonucleotides. According to the procedure used to make this determination, single-stranded probe oligonucleotide radiolabeled at the 5xe2x80x2-end was contacted with rRNA from C. albicans in the presence or absence of helper oligonucleotides. Probe molecules hybridizing the rRNA to form double-stranded hybrids were separated from single-stranded probe molecules by hydroxyapatite capture. The double-stranded hybrids bound to the hydroxyapatite and were detected and quantitated by scintillation counting. The extent of hybridization was then calculated as a percentage. The Tm of the probe:target hybrid advantageously was significantly increased in the presence of one or more helper oligonucleotides.
The following Example describes the methods used to demonstrate that the CalB2208 probe hybridized rRNA from C. albicans and that this interaction was facilitated by including helper oligonucleotides in the hybridization mixture.